Transformation of mycobacterial species using hygromycin resistance as selectable marker

Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10(3)-10(5) transformants (micrograms DNA)-1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.